Functional rescue of a constitutively desensitized beta2AR through receptor dimerization.
We have recently demonstrated that wild-type beta2-adrenergic receptors (beta2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant beta2AR (C341Gbeta2AR), we sought to study whether physical interactions between mutant and wild-type beta2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gbeta2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gbeta2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type beta2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type beta2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A beta2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.
Hebert
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Centre de recherche, Institut de cardiologie de Montréal et Département d'Anésthesie-Réanimation, Université de Montréal, 5000 rue B-elanger est, Montr-eal, PQ, Canada H1T 1C8.
Loisel
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TP
Adam
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L
Ethier
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N
Onge
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SS
Bouvier
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D017354:Point Mutation; D011485:Protein Binding; D011940:Receptor Aggregation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry*; D011994:Recombinant Proteins; D015398:Signal Transduction; D018411:Spodoptera; D013329:Structure-Activity Relationship
D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases